The present invention relates to a method of making covalently bound conjugates of bioactive compounds with biologically site-specific compounds. The bioactive compounds are cosmetic, treatment or therapeutic compounds, such as drugs, hormones, cytotoxic compounds, and the like; the site-specific compounds are desirably antibodies, and particularly monoclonal antibodies and the like. The conjugates from this method retain the bioactive and site-specific properties of the reactants.
It also relates to the chemical compounds which are the product of such methods, and particularly to compounds which are covalently bound conjugates of bioactive molecules and site-specific proteins. In very particular terms it relates to highly homogeneous conjugates with high levels of bioactive action and site specificity and a minimum of side effects and systemic effects in host organisms when administered.
This invention also relates to the method of treating a host organism with such conjugates for the purposes of medical care or serious cosmetic needs of the individual.
In medical treatment of patients, a paramount problem is that powerful prospective drugs can not be used because their side effects on the rest of the body of the patient are too damaging. A great number of chemical compounds, both natural and synthetic, show initial promise as possible new drugs for a wide variety of diseases. Frequently, initial screening tests of a chemical will show good toxicity, growth regulatory effects, immune stimulation or suppression, or other indications of useful properties in vitro. However, when tests are conducted on living organisms, the latent benefit of a large majority of these potential drugs is never realized or severely limited by side effects manifested in vivo. Often, the problem is one of dosage. The required amount or dose of the drug which exhibits the desired therapeutic effect is too high to be tolerated by the organism being treated. This is particularly evident in chemotherapy for cancer in man and other animals. Severe side effects generally accompany the anticancer treatment. Many drugs with even stronger anticancer activity than those presently in use are available, but under normal cytotoxic use regimens, the use of these drugs would kill the patient faster than the cancer.
Many approaches have been tried to circumvent the toxicity problem of strong drugs. The most attractive among these involves the use of chemical conjugates as a means of targeting the drug specifically to the site of action. The goal is to form a conjugate by reacting a drug with a site-specific compound and to use the conjugate for its combination of therapeutic and site-specific properties. The result of effective specific targeting is that it would allow a small dose of conjugate to create a high concentration of the drug in the cancer cells, parasitic organisms, or secretory cells, for example, while keeping the general body burden low. Targeting means which have shown some initial success, and which probably have the greatest potential, are biologically site-specific proteins, especially the monoclonal antibodies and antibody fragments. In their original state, these will bind to particular antigens. The potential advantages of conjugates containing selected monoclonal antibodies and an illustration of the therapeutic effect of these conjugates on cancer cells are described in articles by Yang, H. M. and Reisfeld, R. A., Proc. Natl. Acad. Sci. USA, vol. 85, pps. 1189-1193, February, 1988. and Yang, H. M. and Reisfeld, R. A., J. National Cancer Institute, Vol. 80, No. 14, pps. 154-159, September, 1988.
When prepared by proper screening techniques, monoclonal antibodies possess exquisite specificity for their targets. A target of great medical importance is solid human tumor antigen; these tumors are very resistant to treatment with chemotherapeutic drugs. There are several monoclonal antibodies that are known to be site-specific for these tumors.
The major problems in attempts to achieve therapeutic and site-specific drug compositions arise from the preparation of the drug-antibody conjugate. Traditional chemical methods to prepare conjugates by attaching drugs and related materials to antibodies, either directly or via a linker molecule, suffer from four main problems: namely, that the linkage reaction occurs randomly on active groups of the antibody molecule, frequently with active groups that are near or in the site-specific regions of the molecule, resulting in seriously altered or reduced specificity in the conjugate; the linkage is ordinarily partially labile under physiological conditions, resulting in hydrolysis which causes at least some systemic release of the drug before it reaches its target; the ratio of reagents and the stoichiometry of reagents for control of the conjugation reaction is very difficult to achieve in aqueous solutions since most activating agents that are used for the drugs are degraded by water; and, lipophilic drugs are very difficult, or impossible to link to antibodies to form homogeneous conjugates.
The biologically site-specific compound depends upon the presence of one or more active binding sites in its molecule for the crucial ability to bind only with its target antigen. This is best exemplified by antibodies. It can be seen that the traditional methods for conjugating therapeutic compounds to antibodies show selectivity only in respect to reacting with particular reactive groups on the amino acids which make up the antibody, the .epsilon.-amino group of lysine for example, but no specificity regarding the location of the amino acid in the antibody structure. Since the amino acids are distributed in a near random manner within the antibody structure, the conjugation reactions will proceed with reactive groups that are found within the active binding site of the variable regions as well as those in the constant regions of the molecule. Antibody conjugate preparations generated by traditional methods contain a proportion of molecules which have been inactivated or have had their specificity altered because of bioactive compounds conjugated to the antibody within the active binding site. The percentage of antibody molecules with reduced ability to bind the desired target is related to the number and location of reactive groups within the binding site and the reaction conditions employed. If, for example, a single reactive group within the binding site of the antibody was positioned such that a drug conjugated to the antibody through that reactive group prevented all binding to the target antigen, then exhaustive conjugation of an antibody solution could occur and yet result in a preparation with no target specificity. The inevitable result if no adjustments are made is a preparation with reduced specific activity and increased general toxicity. European Patent Application 0 175 617, J. W. Goers et al. describes the problems which have been encountered in this field by reason of loss of site-specificity in the antibody during the reactions with therapeutic compounds. Similar problems in the preparation of platinum based chemotherapeutics are discussed in European Patent Applications nos. 0 167 310 and 0 169 645 by J. G. Hefferman.
The present invention solves each of the four problems mentioned above to yield compositions of bioactive agent-antibody conjugates which are more stable, more homogeneous, more specific than conjugates prepared by other means, have greater specific activity toward target cells, have fewer by-products and which give fewer side-reactions and thus have lower toxicity toward the patient.
The present invention includes conjugates of antibodies, and particularly monoclonal antibodies with bioactive compounds. These conjugates exhibit or provide the bioactivity of the one parent compound and the site-specificity of the other parent compound. The term bioactive compound is employed herein to include cosmetic compounds and therapeutic compounds such as pharmaceuticals, toxins, cytotoxins, alkylating agents, enzymes, antibiotics, antimetabolites, hormones, neurotransmitters, radioopaque dyes, radioactive isotopes, fluorogenics, bio-markers, lectins, photochemicals, cell membrane modifiers, antiproliferatives and heavy metals. These compounds are used for cosmetic and therapeutic purposes and are typically drugs, particularly cytotoxic and cytoactive compounds and substances including radionuclides, diagnostics, such as radiotracers, fluorescing compounds, and the like, hormones, weight gain and weight loss compounds and other bioactive proteins and related biologically produced compounds and the like which are not themselves site specific in their action. In terms of cellular or host organism effect, the bioactive compound is an agent adapted for functions such as cell destruction, prevention of cell proliferation, hormone therapy, gene therapy, diagnosis of cell condition, tracing of cell location, identification of cellular masses, treatment of healthy cells and modification of healthy cells.
Among the applications for the bioactive, site-specific conjugates which utilize the functions described above are: cancer therapy, treatment of rubella, gram negative infections, hepatitis B, blood clots, tetanus, cold infections, rheumatoid arthritis, cardiovascular disease and septic shock. They can also be used in preventing transplant rejection, for renin inhibition, as pro-drugs, non-therapeutic diagnostics and metal ligating compounds.
The term biologically site-specific compound is employed herein to include proteins, antibodies, antibody fragments, liposomes, viruses, phages and steroid hormones. These compounds are typically water soluble or water dispersible and have at least one active binding site that will attach to individual and particular antigens. The antigen will be uniquely characteristic, or nearly so, of the cell or cellular organism to which the therapeutic treatment or bioactive function is directed. It is to be understood that the site-specific compound will have suitable functional groups for forming covalent bonds with the bioactive compound and that these covalent bonds will be located at a location designated as the conjugation site of the site-specific compound. In addition, the active binding site will refer to those locations where the site-specific compound binds to its target molecule or antigen.
The term bioactive, site-specific conjugate is used herein to refer to the product of condensation between the bioactive compound and the site-specific compound and which conjugate exhibits itself or by controlled hydrolysis provides the bioactivity and site-specificity of the parent compounds. At a molecular level there is at least one covalent bond between the two parent compounds which is formed during the condensation reaction. The conjugate may have a stoichiometry such that the ratio of bioactive compound to site-specific compound is less than one. Typically, there will be two or more moles of bioactive compound per mole of site-specific compound.
A particularly important class of bioactive compounds are the powerful cytotoxic and cytoactive compounds which have been excluded from serious investigation and application in the context of conjugates with monoclonal antibodies and other proteins because these compounds are highly lipophilic, soluble in body fat and insoluble in water. There are a substantial number of such compounds of interest, but efforts to conjugate them with monoclonal antibodies and the like have generally resulted in irreversible denaturation of the proteins in the attempt so that the conjugate has no site-specificity. Typical and representative of these compounds are the carotenoids and retinoids.
Carotenoids and retinoids are closely related compounds which are found in a variety of chemical derivatives in most life forms, including humans. Though their natural functions are poorly understood, these families of chemical compounds, particularly the retinoids, have been shown to have effects relating to cellular differentiation. A variety of retinoids have been shown to stimulate neoplastic cells of various types to differentiate into a nonproliferative form. Most experimental evidence is from in vitro systems. Investigations on animals and humans and development of the retinoids and carotenoids as drugs has been hindered by the toxicity of the compounds at efficacious concentrations. Topical applications of various retinoid preparations have been clearly shown to reverse malignant and premalignant skin lesions. The preponderance of data seem to indicate that carotenoids and retinoids have great potential as anticancer agents, at least for cancers of ectodermal origin, and perhaps more diverse pharmacological applications. The primary problems appear to be that specific effects are difficult to ascertain clinically, and retinoids (and perhaps derivatized carotenoids) are toxic at blood levels sufficient to elicit a positive result. Without a method of effectively targeting these molecules in the form of conjugates, they will remain limited to dietary supplements of ill defined effect and topical agents of very limited use.
Because of the very strong lipophilic properties of the retinoids and carotenoids, it has been virtually impossible to carry out chemical reactions that would successfully link them to monoclonal antibodies or other proteins and produce therapeutic, site-specific conjugates containing the retinoid or carotenoid. Using the novel techniques of the present invention for attaching bioactive compounds to antibodies it is possible to make novel retinoid-antibody conjugates. These conjugates can be prepared by attaching the compound directly to the antibody or by inserting a linker molecule between the two. The linkage between the retinoid and either the linker molecule or the antibody can be either labile or not, depending on what is desired for a particular investigation or other therapeutic purpose. Carotenoid-antibody compounds can be prepared in the same manner and will provide a way to investigate and apply the effects of these compounds and their derivatives.
Antibodies, both poly and monoclonal, are highly preferred compounds for site-specific targeting vehicles in conjugates. Their high specificity of target, recent advances in their mass production, and their documented efficacious use against cancers in various experimental and clinical tests would make them the targeting method of choice. Specifically targeting carotenoids and retinoids at cancer cells would create high concentrations at the target sites without concomitant high blood serum levels and risk of systemic effects. Actual effects of site-specific conjugates of retinoids and carotenoids on the target cancer cells could be ascertained without interference from the liver's regulatory mechanism and subsequent toxicity. New derivatives of the retinoids and carotenoids can be prepared and examined for improved therapeutic effect and new types of cancers can be targeted for site-specific, therapeutic attack. The problem is that conventional methods for conjugating antibodies to drugs do not work for the carotenoids or retinoids. These molecules are very insoluble in aqueous solutions, while antibodies are easily denatured in organic solutions.
The present invention includes the discovery that multiphase reaction systems with interfacial condensation can produce a therapeutic and site-specific conjugate from cytotoxic, and particularly lipophilic cytotoxic compounds, typified by carotenoid and retinoid compounds, and antibodies. The production of these conjugates is made possible by the techniques described herein. It also includes the discovery that this is a process for preparation of bioactive, site-specific conjugates which is highly efficient and improves the yield of the desired conjugate products while simplifying the purifications used to make the compositions pharmaceutically acceptable. It has the advantage that it reduces the formation of by-products and the occurrence of side reactions which have attended and seriously compromised other attempts to prepare successful therapeutic, site-specific conjugates. It provides a substantial improvement in the homogeneity of the desired bioactive, site-specific conjugates. For these reasons, the products of the process will have fewer side effects when used in therapeutic treatment of target cells. And, the natural body response to chemical modifications will be substantially less because of the improved homogeneity of the products. Thus, not only can new and improved bioactive, site-specific conjugates be obtained but also there is a significant improvement in the processes for the preparation of bioactive, site-specific conjugates in general.
It is an object of the present invention to provide a method of conjugating biologically site-specific compounds with bioactive compounds to form covalent bonds between them by the use of interfacial condensation reactions.
It is another object of the present invention to provide a method of conjugating biologically site-specific compounds with bioactive compounds in such a way that the active binding site of the site-specific compound is protected and this active binding property is exhibited by the conjugate along with the bioactive effect.
It is a further object of the present invention to provide a method of conjugating biologically site-specific compounds with bioactive compounds which employs interfacial condensation and where the physical phases for the respective compounds are selected to maximize a chemically favorable environment for each compound.
It is yet another object of the present invention to provide a method of conjugating biologically site-specific compounds with bioactive compounds in which the covalent bonding between the compounds can include a group adapted to provide selected atomic spacing between the compounds or preferential reaction sites for covalent bonding on the compounds and optionally a physiologically cleavable group adapted to release the bioactive compound at the selected site of the target antigen.
It is an object of the present invention to provide a method for preparing bioactive, site-specific conjugates by interfacial condensation in which the bioactive compound is modified by reaction with a spacing or linking or physiologically cleavable group prior to condensation with the biologically site-specific compound.
Another object is the provision of the bioactive, site-specific conjugates which are the products of the processes of the present invention.
Yet another object is the provision of bioactive, site-specific conjugates of bioactive compounds with proteins, particularly with antibodies, and still more particularly with monoclonal antibodies.
Still another object is the covalently bound bioactive, site-specific conjugates of lipophilic bioactive compounds and monoclonal antibodies and other proteins.
A further object is the treatment of host organisms with the conjugates of the present invention.